PROJECT SUMMARY/ABSTRACT In this research proposal we aim to address the question how DNA replication stress causes chromosome fragility, which is one of the underlying mechanisms of genome instability and cancer development. Chromosome fragile sites are effectively preferred sites of DNA double strand breaks inducible upon replication stress, and are an intrinsic and vulnerable feature of our genome. Yet, the locations of the majority of these break sites still remain unknown due to the shortage of suitable genome scale detection methods. Moreover, it is also a long-standing question why different replication inhibitors as well as different cell types produce distinct spectra of breakage formation. We hypothesize that replication inhibitors simultaneously destabilize replication forks and elicit unique patterns of gene expression, thus causing unscheduled clash between replication and transcription and ultimately DNA strand breaks. We propose to test this hypothesis by using a combination of approaches in a yeast model and in human cell lines. We will first take advantage of the genetically amenable yeast model to directly test our core hypothesis. We will learn how the interplay between replication and transcription dynamics impacts DSB formation and genome stability. We will then systematically map drug-specific CFS formation in both yeast and human genomes. These experiments will enable the identification of genomic features associated with DSBs and potentially disease- associated genes such as tumor suppressors. We will also use an established recombination system in the yeast model to test the functionality of the genomic features associated with DNA breaks. Our research will fill the gap in our knowledge of the locations of chromosome fragile sites, enable the discovery of new cancer- associated genes and provide insights into the mechanisms of genome instability. The proposed project encompasses molecular biology, yeast genetics, mammalian cell culture, genomics and bioinformatics. The research team led by Dr. Wenyi Feng includes one predoctoral trainee (second year in a Ph.D. program) and two to three undergraduate trainees (through the summer research fellowship program provided by Upstate Medical University and a credited one to two semester research course provided by Syracuse University), one research support specialist, and one future recruit of a postdoctoral trainee. The predoctoral trainee is at entry level to yeast genetics and mammalian cell culture manipulations and is expected to complete training within the duration of this project. Training for the undergraduates who typically have minimal experimental skills is expected to last one to two years per trainee. Finally, the postdoctoral trainee to be recruited is expected to have experience in mammalian cell culture and/or next-generation sequencing and is also expected to complete training within the duration of this project.